Process and substances for the release of a growth-regulating factor from endothelial cells

ABSTRACT

A method of stimulating endothelial cells cultivated in a nutrient medium to release a growth-factor into the nutrient medium by addition of at least one pentacyclic oxindole alkaloid into the nutrient medium.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of application Ser. No. 09/788,888,filed Feb. 20, 2001, which was a continuation-in-part of applicationSer. No. 09/341,607, which was a continuing application, under 35 U.S.C.§ 120, of International application PCT/AT98/00008, filed Jan. 20, 1998;the application also claims the priority, under 35 U.S.C. § 119, ofAustrian patent application No. A 73/97, filed Jan. 20, 1997; the priorapplications are herewith incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

It is known that pentacyclic oxindole alkaloids exert pharmacologicaleffects on the immune system. Increased phagocytosis of granulocytes [H.Wagner, Kreutzkamp B., Jurcic K., (1985) Planta Med. 51, 419-423] andmoderate inhibition of proliferation of leukemic cells [Stuppner H.,Sturm S., Geisen G., Zillian U., Konwalinka G. (1993) Planta Med. 59,Suppl. A 583] have been demonstrated. A slight but significantlymphocytosis was observed in probands who had taken orally analkaloid-containing extract of the root of Uncaria tomentosa (Willd.)DC. [Keplinger U. (1995) in Krallendorn: Extract from Radix Uncariaetomentosae (Willd.) DC., Information for physicians and pharmacists;Immodal Pharmaka GmbH, 3rd edn.]. From these findings it was deducedthat pentacyclic oxindole alkaloids have immunostimulating orimmunomodulating properties. Patents concerning this were granted [U.S.Pat. No. 5,302,611, WO 86/00524].

It is known that tetracyclic oxindole alkaloids act on the centralnervous system, produce negatively chronotropic and negatively inotropiceffects [Kanatani H., Kohda H., Yamasaki K., Hotta I., Nakata Y., SegawaT., Yamanaka E., Aimi N., Sakai S. I. (1984) J. Pharm. Pharmacol. 37,401-404; Zhang W., Liu G. X. (1986) Act. Pharmacol. Sinica 7 (5),426-428; Zhu Y., Guoxiong H. X. (1993) Chin. J. Pharmacol. Toxicol. 7(2), 117-121], block Ca2+ transport [Sun A., Liu G., Wang X., Zhang W.,Huang X. (1988) Chin. J. Pharmacol. Toxicol. 2 (2), 93-97; Zhang W., LiuG., Huang X. (1987) Act. Pharmacol. Sinica 8, 425-429], and inhibit theaggregation of blood platelets [Jin R. M., Chen C. X., Li Y. K., Xu P.K. (1991) Act. Pharmaceut. Sinica 26 (4), 246-249; Chen C. X., Jin R.M., Li Y. K., Zhong J., Yue L., Chen S. C., Zhou J. Y. (1992) Act.Pharmacol. Sinica 13 (2), 126-130].

It is also known that oxindole alkaloids undergo isomerization insolution. Only recently an analysis of the kinetics of the isomerizationwas reported [Laus G., Brössner D., Senn G., Wurst K. (1996)J.Chem.Soc., Perkin Trans. 2, 1931-1936]. The production of definedmixtures of isomers is known from U.S. Pat. No. 5,723,625. The alkaloidsused in this work were isolated from the roots of Uncaria tomentosa. Thealkaloid content of a number of these plants was investigated. It wasfound that two chemotypes of Uncaria tomentosa occur in nature. Onechemotype of Uncaria tomentosa contains mainly the tetracyclic oxindolealkaloids rhynchophylline and isorhynchophylline, the other one containsthe pentacyclic oxindole alkaloids pteropodine, isopteropodine,speciophylline, uncarine F, mitraphylline and isomitraphylline.Accordingly, they are designated as tetracyclic alkaloid-type orpentacyclic alkaloid-type [Laus G., Brössner D., Keplinger K. (1997)Phytochemistry 45, 855-860]. Transitional forms have also been found insome instances which contain both types of alkaloids in various ratios[Laus G., Keplinger D. (1994) J. Chromatogr. A 662, 243-249]. Thereforethe tetracyclic as well as the pentacyclic alkaloids were used in theinvestigations which are described in the following.

General Structure of Pentacyclic Oxindole Alkaloids with Notation ofStereochemistry:

1 Pteropodine 3S, 7R, 15S, 19S, 20S 2 Isopteropodine 3S, 7S, 15S, 19S,20S 3 Speciophylline 3R, 7S, 15S, 19S, 20S 4 Uncarine F 3R, 7R, 15S,19S, 20S 5 Mitraphylline 3S, 7R, 15S, 19S, 20R 6 Isomitraphylline 3S,7S, 15S, 19S, 20R

General Structure of Tetracyclic Oxindole Alkaloids with Notation ofStereochemistry:

7 Rhynchophylline 3S, 7R, 15S, 20R 8 Isorhynchophylline 3S, 7S, 15S, 20RExperiments in Cell Cultures

The effect of the alkaloids was studied in endothelial cells becausethey are known for interactions with immunologic reactions [Kirchner H.,Kruse A., Neustock P., Rink L. (1993) Cytokine und Interferone:Botenstoffe des Immunsystems, 61]. It was recognized that thepentacyclic alkaloids (c=1 μM) induced transformed EA.hy926 endothelialcells [Edgell C. -J. S., McDonald C. C., Graham J. B. (1983) Proc. Natl.Acad. Sci. USA 80, 3734-3737] as well as normal human umbilical veinendothelial cells (HUVEC, ATCC CRL-1730) to release a factor into theculture medium which significantly affects the proliferation oflymphocytes. In general, RPMI-1640 was used as the culture medium forEA.hy926 endothelial cells and lymphocytes, completed with 10% fetalcalf serum, 2 mM glutamin, 50 units/ml penicillin G and 50 μg/mlstreptomycin. For human umbilical vein endothelial cells HAM F12(Sigma-Aldrich Company, St. Louis, USA) was used as the culture medium,completed with 10% fetal calf serum, 60 μg/ml Endothelial Cell GrowthSupplement and 100 μg/ml heparin.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1: Qualitative differentiation of the two chemotypes of Uncariatomentosa by thin-layer-chromatography. Columns 1 and 2 are results fromacid and alkaline solutions of Uncaria tomentosa containing pentacyclicoxindole alkaloids (IMM-2414), columns 3 and 4 are results from acid andalkaline solutions of Uncaria tomentosa containing tetracyclic oxindolealkaloids (IMM-2418), and colums 6 and 7 show results from acid andalkaline mixtures of both. Column 5 is a reference solution.

FIG. 2: Separation of pentacyclic and tetracyclic oxindole alkaloids byHPLC.

FIG. 3: Equilibria of isomerization Kaq, Korg and partition Korg/aq oftwo isomers (1) and (2) in a two-phase-system,where${K_{\quad{org}}\left( 1\rightarrow 2 \right)} = {\frac{K_{{org}/{aq}}(2)}{K_{{org}/{aq}}(1)}{{K_{aq}\left( 1\rightarrow 2 \right)}.}}$

FIG. 4: pH-dependence of equilibrium concentrations (in mol-%) in anoctanol-water system containing mitraphylline and isomitraphylline as anexample.

FIG. 5: Increase of proliferation of normal human B lymphocytes,stimulated by supernatants of EA.hy926 cells which were grown in thepresence of 1 μM IMM-2414, IMM-2417, IMM-2418.

FIG. 6: Inhibition of the proliferation of Jurkat cells (ATCC E6.1),treated with supernatants of EA.hy926 cells which were grown in thepresence of 1 μM IMM-2414, IMM-2417, IMM-2418.

FIG. 7: Inhibition of the proliferation of highly activated human B andT lymphocytes (lymphoblasts) treated with supernatants of EA.hy926 cells(SN) which were grown in the presence of 1 μM IMM-2414.

FIG. 8: Inhibition of the proliferation of highly activated human Tlymphocytes (lymphoblasts) treated with the alkaloids IMM-2414,IMM-2417, IMM-2435 or with supernatants of EA.hy926 cells (SN) whichwere grown in the presence of 1 μM IMM-2414, IMM-2417, IMM-2435.

FIG. 9: Proliferation of normal human T lymphocytes, treated withIMM-2414 and/or IMM-2418, or with supernatants of EA.hy926 cells (SN)which were grown in the presence of 1 μM IMM-2414 and/or IMM-2418.

FIG. 10: Antagonistic effect of tetracyclic oxindole alkaloids (TOA) onthe biological activity on Raji and Jurkat cells caused by pentacyclicoxindole alkaloids (POA).

DETAILED DESCRIPTION OF THE INVENTION

Thin-layer Chromatographic Identification Test:

Thin layer chromatography provides an excellent method for the test ofidentity of the drug, especially when the characteristic pH-dependentisomerization behavior of the oxindole alkaloids is taken as anadditional criterion. Ajmalicine is proposed as a reference substancebecause of its similar structure and commercial availability. In orderto compensate for variations in chromatographic conditions the Rf valuesare referred to ajmalicine (hRajmalicine values, Table 1). TABLE 1hR_(ajmalicine) values of the alkaloids Alkaloid hR_(ajmalicine)Speciophylline 12 Mitraphylline 26 Uncarine F 56 Isomitraphylline 75Pteropodine 83 Isopteropodine 95 Rhynchophylline 22 Isorhynchophylline91 Ajmalicine 100

Test solutions: 1 g of the drug is heated with 50 ml distilled water for45 minutes at 85° C. The extract is decanted and the drug is washed with20 ml water. The combined extracts are divided into two portions. Oneportion is acidified by the addition of 1 drop of hydrochloric acid 7%(approx. pH 4, solutions 1, 3 and 6) and refluxed for 24 h, the secondportion is made alkaline with 1 drop of sodium hydroxide solution 8.5%(approx. pH 8, solutions 2, 4 and 7) and maintained at 50° C. for 24 h.Afterwards 2 drops of sodium hydroxide solution 8.5% are added to theacidic solution. All solutions are extracted with 3×5 ml chloroform,collecting at least 4 ml of the organic layer in each extraction step.The extracts are dried by the addition of anhydrous sodium sulfate andthe solvent is evaporated. The residues are dissolved in 0.5 mlchloroform and the resulting solutions are used for thin layerchromatography. 1 mg ajmalicine (Fluka, Switzerland) is dissolved in 1ml chloroform to give the reference solution (solution 5). Spots of 10μl are applied to TLC-plastic sheets of silica 60 F254 (20×20 cm, 0.2 mmthickness of layer; Merck No. 5735). A mixture of ethyl acetate/n-hexane(95:5) is used to develop the chromatogramme, and fluorescence quenchingat 254 nm is used for detection.

The possible cases are depicted in FIG. 1. In case of the pentacyclicalkaloid-type U. tomentosa spots of six alkaloids are observed.Speciophylline, mitraphylline and pteropodine dominate in solution 1which was isomerized in acid (column 1), while isopteropodine andisomitraphylline prevail in solution 2 which was isomerized in alkali(column 2). The undesired alkaloids rhynchophylline andisorhynchophylline from tetracyclic alkaloid-type U. tomentosa can beseen clearly and exhibit similar dependance on the pH of the isomerizedsolutions 3 and 4 as mitraphylline and isomitraphylline. Column 5 is thereference compound ajmalicine. Columns 6 and 7 indicate typical drugmixtures. An HPLC chromatogramme which allows the analysis ofpentacyclic and tetracyclic oxindole alkaloids is shown in FIG. 2.Method: LiChroCART 125 mm×4 mm (I.D.) columns packed with LiChrospher100 RP-18 (5 μm) (Merck), thermostatted at 52° C., acetonitrile-aqueousphosphate buffer pH7 (40:60) with a flow of 1.3 ml/min. Detection at 247nm.

Composition of Alkaloid Mixtures Used

As oxindole alkaloids undergo isomerization in aqueous solution, nosingle isomers but groups of isomers were employed. First, a mixture ofpentacyclic alkaloids (IMM-2414) was used, then the isomer groups ofmitraphylline (IMM-2417), rhynchophylline (IMM-2418) and pteropodine(IMM-2435) were used. The composition of the mixtures is given in Table2. The composition of the alkaloid mixtures was determined by HPLCanalyses. TABLE 2 Composition of alkaloid mixtures used Code IMM-Alkaloid IMM-2414 IMM-2417 IMM-2418 2435 Speciophylline 4% — —  4%Uncarine F 6% — —  6% Pteropodine 28%  — — 30% Isopteropodine 57%  — —60% Mitraphylline 2% 33% — — Isomitraphylline 3% 67% — — Rhynchophylline— — 40% — Isorhynchophylline — — 60% —Comments:The percent quotations are percents by weight.The composition of the alkaloid mixtures was determined by HPLCanalysis.

Simple derivatives were also used: the alkaloid carboxylic acids(IMM-2413) prepared by alkaline hydrolysis of the alkaloid mixture(IMM-2414), and the alkaloid N-oxides (IMM-2433) prepared by oxidationof the mixture (IMM-2414) using hydrogen peroxide.

Distribution of the Alkaloids in Biological Systems

Cells in a culture medium can be viewed as a two-phase system consistingof water and lipids. The distribution of the alkaloids in cell culturesand in mixtures of octanol and water was studied. It was found that thevarious isomers behave differently (Table 3). The alkaloids werepartitioned between equal volumes of octanol and aqueous phosphatebuffer pH 7 (0.01 M) at 20° C. The concentrations c of the alkaloidswere determined by HPLC analysis and the coefficients of distribution${K\quad{O/W}} = \frac{c_{({{in}\quad{control}})}}{c_{({{in}\quad{water}})}}$

were calculated. TABLE 3 Common logarithm of the partition coefficientsK O/W at pH 7 Alkaloid log K O/W Pteropodine 2.9 Isopteropodine 3.2Speciophylline 1.4 Uncarine F 3.1 Mitraphylline 2.5 Isomitraphylline 2.8Rhynchophylline 2.7 Isorhynchophylline 3.1

As expected the equilibrium of isomers in a two-phase system (1aq

1org; 2aq

2org, FIG. 3) depends not only on the pH value of the aqueous phase butalso on the amount and nature of the organic phase. For an example, theequilibria of mitraphylline and isomitraphylline in an octanol-watersystem (1:1) at pH values from 3 to 7 are shown (FIG. 4). It can be seenthat in the pH range from 4 to 7 isomitraphylline is predominant in theoctanol phase, with a maximum of 94 mol-% at approximately pH 5.However, below pH 3 mitraphylline begins to prevail in the aqueousphase. At no pH value is isomitraphylline produced in the aqueous phaseto a reasonable extent. Therefore, the distribution of the isomers in a2-phase system is clearly different compared to the situation in apurely aqueous solution.

In EA.hy926 endothelial cell cultures which were incubated with variousalkaloid mixtures (c≈1 μM) a decline of the concentrations ofisopteropodine or isomitraphylline, respectively, was observed after 7days, whereas in contrast the concentration of isorhynchophyllineremained nearly constant (Table 4). A RPMI-1640 culture medium(Sigma-Aldrich Company, St. Louis, USA) completed with 10% by volumefetal calf serum, 2 mM glutamin, 50 units/ml penicillin G, and 50 μg/mlstreptomycin was used. TABLE 4 Change of the alkaloid concentrations(mg/l) in the RPMI-1640 nutrient medium of EA.hy926 endothelial cellcultures after 7 days Stimulant (concentration) Alkaloid Start 7 DaysIMM-2414 (1.0 μM) Isopteropodine 0.22 0.12 Pteropodine 0.10 0.16 Total0.32 0.28 IMM-2417 (1.0 μM) Isomitraphylline 0.28 0.14 Mitraphylline0.10 0.11 Total 0.38 0.25 IMM-2418 (1.1 μM) Isorhynchophylline 0.27 0.27Rhynchophylline 0.15 0.13 Total 0.42 0.40 IMM-2435 (1.4 μM)Isopteropodine 0.35 0.18 Pteropodine 0.11 0.20 Total 0.46 0.38Comments:IMM-2414 = solution of standard mixture of pentacyclic alkaloids inmediumIMM-2417 = solution of mixture of isomers of mitraphylline in mediumIMM-2418 = solution of mixture of isomers of rhynchophylline in mediumIMM-2435 = solution of mixture of isomers of pteropodine in medium

The solubility of the alkaloids in cell membranes does not offer anexplanation for the different changes of concentration. Rather, thedecline in concentration is a consequence of physiological processes inthe cytosol. Compared with pure medium, the isomerization takes adifferent course in the presence of endothelial cells. Within the 7 daysof an experiment (EA.hy926 endothelial cells in RPMI-1640: pH 7.5 at thestart, pH 8.1 after 7 days) an untypical mixture of isomers is formedwhich contains pteropodine and isopteropodine or mitraphylline andisomitraphylline, respectively, in a ratio of approx. 1:1, whereas incontrast rhynchophylline and isorhynchophylline isomerize to give atypical equilibrium mixture in a ratio of 1:2. Of course, activity ofsingle isomers cannot be evaluated in this test model because of theisomerization. But it can be established that a turnover takes place inthe case of the pentacyclic but not tetracyclic alkaloids. The effectsof this factor on lymphocytes were investigated in detail. It was foundthat immortalized cells, e.g. the Epstein-Barr virus-transformedlymphoblastoid cell line Raji or the leukaemic cell line Jurkat, andnormal human B and T lymphocytes (isolated from whole blood of normaldonors) are affected by the factor in different ways:

1. Supernatants (SN) of endothelial cell cultures stimulated withIMM-2414 for 7 days were added to normal human non-activated or weaklyactivated B and T lymphocyte cultures in several concentrations. Anincreased proliferation of the lymphocytes was measured by [3H]thymidineuptake after 5 days (Table 5). Thus, the lymphocytes were treated with 1μCi [3H]thymidine for 18 hours, harvested on nitrocellulose, andradioactivity was measured in a scintillation counter (cpm=counts perminute). Every assay was performed in triplicate. TABLE 5 Proliferation(cpm after [3H]thymidine uptake) of normal human non- activated orweakly activated B and T lymphocytes in med. RPMI-1640 Stimulant(dilution) B lymphocytes T lymphocytes Medium  363 ± 213 349 ± 114IMM-2414 (1 μM)  352 ± 323 332 ± 82  and stimulated with EA.hy926endothelial cell culture supernatants SN-Medium (1:4) 1015 ± 618 591 ±252 SN-2414 (1:4) 1527 ± 540  1242 ± 752*** SN-Medium (1:8) 1381 ± 390493 ± 278 SN-2414 (1:8)   2039 ± 530***   1084 ± 549**** SN-Medium(1:16) 1263 ± 299 371 ± 151 SN-2414 (1:16)  1795 ± 584*  549 ± 250**Comments: Medium = RPMI-1640 completed as specified above IMM-2414 =solution of standard mixture of pentacyclic alkaloids in mediumSN-Medium = supernatant of endothelial cell culture in medium, dilutedwith medium in the ratio given SN-2414 = supernatant of endothelial cellculture stimulated with the standard mixture of pentacyclic alkaloids inmedium for 7 days, diluted with medium in the ratio given Mean values ±standard deviation of at least 7 experiments are given. Significance wasevaluated with Student's t-test for paired samples: *P < 0.05, **P <0.01, ***P < 0.005, ****P < 0.001.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of non-stimulated endothelial cellcultures (SN-Medium) increase the proliferation, and the supernatants ofcells stimulated with IMM-2414 increase the proliferation even more. Themaximum effect was obtained with T lymphocytes at a dilution of 1:8 andwith B lymphocytes at 1:4 of the supernatant SN-2414.

2. Supernatants of endothelial cell cultures (SN-2414) stimulated withIMM-2414 for 7 days and non-stimulated endothelial cell cultures(SN-medium) were added to transformed cells (Raji ATCC CCL86 and JurkatATCC E6.1) in several concentrations. In contrast to the B and Tlymphocytes, an inhibition of proliferation of the transformed cells wasmeasured by [3H]thymidine uptake after 2 days (Table 6). Cultures of themyeloid cell line U937 (ATCC CRL1593.2) were also studied. Thetransformed cells were treated with 0.5 μCi [3H]thymidine for 5 hours,harvested on nitrocellulose, and radioactivity was measured in ascintillation counter (cpm=counts per minute). TABLE 6 Proliferation(cpm after [3H]thymidine uptake) of various cell lines in mediumRPMI-1640 Stimulant (dilution) Raji CCL86 a Jurkat E6.1 a U937 CRL1593.2b Medium 21621 ± 5755 35085 ± 13876 121349 ± 18653 IMM- 21698 ± 629935688 ± 14020 123346 ± 26412 2414 (1 μM) and under the influence ofEA.hy926 endothelial cell culture supernatants SN- 32967 ± 9652 26544 ±17492  86115 ± 30792 Medium (1:2) SN-2414  4801 ± 3766****  4282 ±3186**  84736 ± 33654 (1:2) SN- 30919 ± 11134 34716 ± 17394 101093 ±24813 Medium (1:4) SN-2414  9178 ± 7671****  7163 ± 6268***  94257 ±30331 (1:4) SN- 21976 ± 7443 41428 ± 16648 118634 ± 19980 Medium (1:8)SN-2414 11357 ± 5308*  8044 ± 3921*** 107771 ± 30975 (1:8)Comments:Medium = RPMI-1640 completed as specified aboveIMM-2414 = solution of standard mixture of pentacyclic alkaloids inmediumSN-Medium = supernatant of endothelial cell culture in medium, dilutedwith medium in the ratio givenSN-2414 = supernatant of endothelial cell culture stimulated with thestandard mixture of pentacyclic alkaloids in medium for 7 days, dilutedwith medium in the ratio givenMean values ± standard deviation of at least a 7 experiments, b 3experiments are given.Significance was evaluated with Student's t-test for paired samples:*P < 0.05,**P < 0.01,***P < 0.005,****P < 0.001.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the IMM-2414-stimulatedendothelial cell cultures inhibit the profileration of Raji and Jurkatcells dose-dependently, whereas the myeloid cell line U937 is notaffected.

3. The anti-proliferative effect on Raji and Jurkat cells is not due tocytotoxicity, as shown by unchanged viability of the cells (Table 7).TABLE 7 Viability (in %) of Raji and Jurkat cells after stimulation withIMM- 2414 or supernatants of EA.hy926 endothelial cell cultures whichwere cultivated with IMM-2414 Raji CCL86 Jurkat E6.1 Stimulant(dilution) 1st day 2nd day 1st day 2nd day Medium 93.5 95.1 95.8 93.7IMM-2414 (1 μM) 96.8 90.1 95.2 95.8 and under the influence of EA.hy926endothelial cell culture supernatants SN-Medium (1:2) 95.4 95.3 96.492.0 SN-2414 (1:2) 98.4 92.2 94.4 92.2 Comments: Medium = RPMI-1640completed as specified above IMM-2414 = solution of standard mixture ofpentacyclic alkaloids in medium SN-Medium = supernatant of endothelialcell culture in medium, diluted with medium in the ratio given SN-2414 =supernatant of endothelial cell culture stimulated with the standardmixture of pentacyclic alkaloids in medium for 7 days, diluted withmedium in the ratio given

The viability of the cells was determined by trypan blue exclusion after1 and 2 days of stimulation with IMM-2414. In all cases the viabilitywas higher than 90%.

4. Supernatants of endothelial cell cultures stimulated with IMM-2414,IMM-2417 or IMM-2435 for 7 days (SN-2414, SN-2417, SN-2435) andnon-stimulated (SN-medium) were added to cultures of human highlyactivated T lymphocytes (lymphoblasts) in several concentrations (Table8). TABLE 8 Proliferation (cpm after [3H]thymidine uptake) of humanhighly activated T lymphocytes (lymphoblasts) in medium RPMI-1640Stimulant (dilution) T lymphoblasts Medium 4065 IMM-2414 4557 IMM-24173929 IMM-2435 4124 and under the influence of EA.hy926 endothelial cellsupernatants SN-medium (1:4) 3113 SN-2414 (1:4) 652 SN-2417 (1:4) 2285SN-2435 (1:4) 1887 SN-medium (1:8) 3139 SN-2414 (1:8) 2628 SN-2417 (1:8)1595 SN-2435 (1:8) 2380 SN-medium (1:16) 2320 SN-2414 (1:16) 1852SN-2417 (1:16) 1913 SN-2435 (1:16) 1821 Comments: Medium = RPMI-1640completed as specified above IMM-2414 = solution of standard mixture ofpentacyclic alkaloids in medium IMM-2417 = solution of mixture ofisomers of mitraphylline in medium IMM-2435 = solution of mixture ofisomers of pteropodine in medium SN-medium = supernatant of endothelialcell culture in medium, diluted with medium in the ratio given SN-2414 =supernatant of endothelial cell culture stimulated with the standardmixture of pentacyclic alkaloids in medium for 7 days, diluted withmedium in the ratio given SN-2417 = supernatant of endothelial cellculture stimulated with the mixture of isomers of mitraphylline inmedium for 7 days, diluted with medium in the ratio given SN-2435 =supernatant of endothelial cell culture stimulated with the mixture ofisomers of pteropodine in medium for 7 days, diluted with medium in theratio given. All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the non-stimulated endothelialcell cultures (SN-medium) already inhibit the proliferation, thesupernatants (SN-2414, SN-2417, SN-2435) of endothelial cell culturesstimulated with IMM-2414, IMM-2417, IMM-2435 further enhance this effect(FIG. 8). The dose dependance of the effect is clearly seen.

5. Supernatants of endothelial cell cultures (SN-2412) stimulated withIMM-2414 for 7 days and non-stimulated (SN-medium) were added to highlyactivated B or T lymphocyte cultures (lymphoblasts from peripheral bloodor tonsils) in several concentrations. Table 9 shows an inhibition ofproliferation of the lymphocytes, measured by [3H]thymidine uptake(cpm=counts per minute). TABLE 9 Inhibition of the proliferation (cpmafter [3H]thymidine uptake) of highly activated human B and Tlymphocytes (lymphoblasts) in medium RPMI-1640 Stimulant (dilution) Blymphoblasts T lymphoblasts Medium 17840 2186 IMM-2414 (1 μM) 16610 2097and under the influence of EA.hy926 endothelial cell culturesupernatants SN-Medium (1:8) 9594 1254 SN-2414 (1:8) 1527 884 SN-Medium(1:16) 13865 1554 SN-2414 (1:16) 1699 728 SN-Medium (1:32) 13903 2049SN-2414 (1:32) 2534 720 Comments: Medium = RPMI-1640 completed asspecified above IMM-2414 = solution of standard mixture of pentacyclicalkaloids in medium SN-Medium = supernatant of endothelial cell culturein medium, diluted with medium in the ratio given SN-2414 = supernatantof endothelial cell culture stimulated with the standard mixture ofpentacyclic alkaloids in medium for 7 days, diluted with medium in theratio given All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the non-stimulated endothelialcell cultures (SN-Medium) already inhibit the proliferation, thesupernatants of the IMM-2414-stimulated endothelial cell cultures(SN-2414) further enhance this effect (FIG. 7). The dose dependance ofthe effect is clearly seen.

In another experiment, supernatants of HUVEC endothelial cell culturesstimulated with IMM-2414 for 7 days (SN-2414) and non-stimulated(SN-medium) were added to T lymphocyte cultures in severalconcentrations. The influence on proliferation of the lymphocytes wasmeasured by [3H]thymidine uptake (cpm=counts per minute). The resultsare shown in Table 10. TABLE 10 Proliferation (cpm after [3H]thymidineuptake) of human normal T lymphocytes in medium HAM F12 Stimulant(dilution) T lymphocytes Medium 884 IMM-2414 (2 μM) 885 and under theinfluence of supernatants from HUVEC endothelial cell cultures SN-Medium(1:4) 1339 SN-2414 (1:4) 1887 SN-Medium (1:8) 1106 SN-2414 (1:8) 1509SN-Medium (1:16) 913 SN-2414 (1:16) 1279 Comments: Medium = HAM F12completed as specified above IMM-2414 = solution of standard mixture ofpentacyclic alkaloids in medium SN-Medium = supernatant of endothelialcell culture in medium, diluted with medium in the ratio given SN-2414 =supernatant of endothelial cell culture stimulated with the standardmixture of pentacyclic alkaloids in medium for 7 days, diluted withmedium in the ratio given All values result from single experiments.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the non-stimulated endothelialcell cultures (SN-medium) already increase the proliferation, thesupernatants of the IMM-2414-stimulated endothelial cell cultures(SN-2414) further enhance this effect. The dose dependance of the effectis clearly seen. Thus, activities produced by HUVEC culture supernatantsare somewhat weaker but significant, too.

7. The release of the growth-factor was effected by the groups ofisomers of the pentacyclic alkaloids pteropodine or mitraphylline(IMM-2414, IMM-2417 or IMM-2435), but not by the group of isomers of thetetracyclic alkaloid rhynchophylline (IMM-2418), as can be seen in Table11. Supernatants of endothelial cell cultures stimulated with IMM-2414,IMM-2417 or IMM-2418 for 7 days (SN-2414, SN-2417, SN-2418) andnon-stimulated (SN-medium) were added to cell cultures in severalconcentrations. The influence on proliferation of the cells was measuredby [3H]thymidine uptake (cpm=counts per minute). TABLE 11 Proliferation(cpm after [3H]thymidine uptake) of Jurkat cells (ATCC E6.1) and normalhuman B lymphocytes in medium RPMI-1640 B Stimulant (dilution) JurkatE6.1 a Stimulant (dilution) lymphocytes b Medium 32737 Medium 632IMM-2414 (1 μM) 35688 IMM-2414 (1 μM) 560 IMM-2417 (1 μM) 33700 IMM-2417(1 μM) 606 IMM-2418 (1 μM) 31440 IMM-2418 (1 μM) 501 and stimulated withsupernatants of EA.hy926 cells SN-Medium (1:2) 21673 SN-Medium (1:8)1639 SN-2414 (1:2) 4282 SN-2414 (1:8) 2082 SN-2417 (1:2) 3953 SN-2417(1:8) 2183 SN-2418 (1:2) 15724 SN-2418 (1:8) 1908 SN-Medium (1:4) 25288SN-Medium (1:16) 1306 SN-2414 (1:4) 7163 SN-2414 (1:16) 1617 SN-2417(1:4) 6068 SN-2417 (1:16) 2289 SN-2418 (1:4) 26132 SN-2418 (1:16) 1474SN-Medium (1:8) 28124 SN-Medium (1:32) 1231 SN-2414 (1:8) 8044 SN-2414(1:32) 1505 SN-2417 (1:8) 11783 SN-2417 (1:32) 2258 SN-2418 (1:8) 30190SN-2418 (1:32) 1437 Comments: Medium = RPMI-1640 completed as specifiedabove IMM-2414 = solution of standard mixture of pentacyclic alkaloidsin medium IMM-2417 = solution of mixture of isomers of mitraphylline inmedium IMM-2418 = solution of mixture of isomers of rhynchophylline inmedium SN-medium = supernatant of endothelial cell culture in medium,diluted with medium in the ratio given SN-2414 = supernatant ofendothelial cell culture stimulated with the standard mixture ofpentacyclic alkaloids in medium for 7 days, diluted with medium in theratio given SN-2417 = supernatant of endothelial cell culture stimulatedwith the mixture of isomers of mitraphylline in medium for 7 days,diluted with medium in the ratio given SN-2418 = supernatant ofendothelial cell culture stimulated with the mixture of isomers ofrhynchophylline in medium for 7 days, diluted with medium in the ratiogiven a Mean values of at least 3 parallel experiments. b Results ofsingle experiments.

The alkaloids alone do not have an effect compared to the blank medium.The supernatants of the non-stimulated endothelial cell cultures(SN-medium) already inhibit the proliferation of the Jurkat cells (FIG.6) and increase the proliferation of the B lymphocytes (FIG. 5). Thesupernatants of the IMM-2414 and IMM-2417-stimulated endothelial cellcultures (SN-2414, SN-2417) further enhance this effect. The dosedependance of these effects are clearly seen. It is to notice thatsupernatants of endothelial cell cultures stimulated with IMM-2417increase the proliferation of B lymphocytes even when diluted in theratio 1:16 or 1:32, whereas the activity of endothelial cell culturesstimulated with IMM-2414 already decreases (SN-2414). The supernatantsof the IMM-2418-stimulated endothelial cell cultures (SN-2418) produceno effect compared to supernatants of non-stimulated endothelial cellcultures (SN-medium).

8. Rather it was shown that the tetracyclic alkaloids actantagonistically on the production and/or release of the growth-factor.This could be connected with their known capability of blocking Ca2+transport. Furthermore, they reduce the influence of the factor on theproliferation of T-lymphocytes in a dose-dependent manner (addition of 1pM IMM-2418 to an active supernatant reduces the activity by 10%, 10 μMby 20%). Results are shown in Table 12. Supernatants of endothelial cellcultures stimulated with IMM-2414 and/or IMM-2418 for 7 days (SN-2414,SN-2418, SN-2414/2418 and SN-2414/10×2418) and non-stimulated(SN-medium) were added to T lymphocyte cultures in severalconcentrations. The influence on proliferation of the lymphocytes wasmeasured by [3H]thymidine uptake (cpm=counts per minute). TABLE 12Proliferation (cpm after [3H]thymidine uptake) of human normal Tlymphocytes in medium RPMI-1640 Stimulant (dilution) T lymphocytesMedium 598 ± 429 IMM-2414 (1 μM) 590 ± 420 IMM-2418 (1 μM) 564 ± 409IMM-2414 (1 μM)/2418 (1 μM) 536 ± 416 IMM-2414 (1 μM)/2418 (10 μM) 602 ±504 and under the influence of EA.hy926 endothelial cell supernatantsSN-Medium (1:4) 1900 ± 1603 SN-2414 (1:4) 2694 ± 1662 SN-2418 (1:4) 1956± 1618 SN-2414/2418 (1:4) 1890 ± 1712 SN-2414/10 × 2418 (1:4) 1479 ±1191 SN-Medium (1:8) 1581 ± 1448 SN-2414 (1:8) 2144 ± 1402 SN-2418 (1:8)1588 ± 1393 SN-2414/2418 (1:8) 1698 ± 1644 SN-2414/10 × 2418 (1:8) 1692± 1422 Comments: Medium = RPMI-1640 completed as specified aboveIMM-2414 = solution of standard mixture of pentacyclic alkaloids inmedium IMM-2418 = solution of mixture of isomers of rhynchophylline inmedium SN-Medium = supernatant of endothelial cell culture in medium,diluted with medium in the ratio given SN-2414 = supernatant ofendothelial cell culture stimulated with the standard mixture ofpentacyclic alkaloids in medium for 7 days, diluted with medium in theratio given SN-2418 = supernatant of endothelial cell culture stimulatedwith the mixture of isomers of rhynchophylline in medium for 7 days,diluted with medium in the ratio given SN-2414/2418 = supernatant ofendothelial cell culture stimulated with the standard mixture ofpentacyclic alkaloids and the mixture of isomers of rhynchophilline inequal parts in medium for 7 days, diluted with medium in the ratio givenSN-2414/10 × 2418 = supernatant of endothelial cell culture stimulatedwith the standard mixture of pentacyclic alkaloids and the mixture ofisomers of rhynchophilline (1:10) in medium for 7 days, diluted withmedium in the ratio given. The values represent mean values + standarddeviation of 7 experiments.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the non-stimulated endothelialcell cultures (SN-medium) already increase the proliferation, thesupernatants of the IMM-2414-stimulated endothelial cell cultures(SN-2414) further enhance this effect. The supernatants of the IMM-2418stimulated endothelial cell cultures (SN-2418) do not have an effectcompared to the supernatants of non-stimulated endothelial cell cultures(SN-medium). The supernatants of the IMM-2414 and IMM-2418-stimulatedendothelial cell cultures (SN-2414/2418) do not have an effect, either,compared to the supernatants of non-stimulated endothelial cell cultures(SN-medium). IMM-2418 therefore cancels the effect of IMM-2414 (FIG. 9).Highly concentrated (diluted 1:4) supernatants of endothelial cellcultures stimulated with tenfold concentrated IMM-2418 (SN-2414/10×2418)produce of slight inhibition of proliferation compared to supernatantsof non-stimulated cultures (SN-medium).

9. Admixture of 0.01, 0.1 and 1 μM tetracyclic oxindole alkaloids to 1μM pentacyclic oxindole alkaloids (pteropodine isomers as well asmitraphylline isomers) as stimulant reduced the effect of thesupernatants on Raji and Jurkat cells in a dose-dependent manner.Supernatants (SN) of endothelial cell cultures stimulated with IMM-2417,IMM-2435 and/or IMM-2418 for 7 days and non-stimulated(SN-medium=control) were added to transformed lymphoblastoid Raji andJurkat cell cultures. The inhibition of proliferation of the cells wasmeasured by [3H]thymidine uptake (cpm=counts per minute). Thetetracyclic oxindole alkaloids act antagonistically on the pentacyclicoxindole alkaloids in a dose-dependent manner (FIG. 10). This could beconnected with their known capability of blocking Ca2+ transport.Furthermore, they reduce the influence of the factor on theproliferation of T-lymphocytes in a dose-dependent manner (addition of 1μM IMM-2418 to an active supernatant reduced the activity by 10%, 10 μMby 20%). In Table 13, the values are given in % of the control(SN-Medium) of the proliferation as effected by the mix-supernatants in3 dilutions (1:4, 1:8, and 1:16). TABLE 13 Proliferation oflymphoblastoid cell lines Raji CCL86 and Jurkat E6.1 after treatmentwith EA.hy936 endothelial cell culture supernatants (% of control ±s.d.) Jurkat E6.1 Raji CCL86 SN-2417 (1 μM)  52 ± 2** 36 ± 6*** SN-[2417(1 μM) + 2418 (0.01 μM)]  74 ± 4** 57 ± 1*** SN-[2417 (1 μM) + 2418 (0.1μM)] 88 ± 3* 73 ± 3**  SN-[2417 (1 μM) + 2418 (1 μM)] 89 ± 19 85 ± 16 SN-2418 (1 μM) 104 ± 1  99 ± 5   SN-2435 (1 μM) 50 ± 2* 32 ± 3***SN-[2435 (1 μM) + 2418 (0.01 μM)] 78 ± 24 53 ± 4*** SN-[2435 (1 μM) +2418 (0.1 μM)] 83 ± 16 67 ± 6**  SN-[2435 (1 μM) + 2418 (1 μM)] 87 ± 2382 ± 16  SN-2418 (1 μM) 113 ± 11  100 ± 5   SN-Medium - control 100 100Comments:SN-Medium = supernatant of endothelial cell culture in medium, dilutedwith medium in the ratio givenSN-2417 = supernatant of endothelial cell culture stimulated with themixture of isomers of mitraphylline in medium for 7 days in theconcentration givenSN-2418 = supernatant of endothelial cell culture stimulated with themixture of isomers of rhynchophylline in medium for 7 days in theconcentration givenSN-2435 = supernatant of endothelial cell culture stimulated with themixture of isomers of pteropodine in medium for 7 days, diluted withmedium in the ratio givenSignificantly different from control (Student's t-test):*p < 0.01,*p < 0.005,***p < 0.001;n = 6.

-   10. Alkaloids alone were added to lymphocytes to exclude the    possibility of a direct effect. Endothelial cells were grown without    alkaloids and the alkaloids were added to the supernatant in order    to prove that stimulation of the cells by the alkaloids is necessary    for the production and/or release of the factor. Both experiments    showed that neither the alkaloids alone nor in combination with a    supernatant of untreated endothelial cells exert an effect on the    proliferation of lymphocytes. Thus it was shown that the pentacyclic    isomers do not affect directly the proliferation but rather induce    endothelial cells to produce and/or release a factor which    influences the proliferation of lymphocytes. It is assumed that    endothelial cells produce a similar factor even without stimulation    because the supernatants of unstimulated cultures do influence the    proliferation, although to a minor degree. A lower dosage of    pentacyclic oxindole alkaloids (0.1 μM) did not induce the    production and/or release of the factor anymore (Table 14).

Supernatants of endothelial cell cultures stimulated with IMM-2414(c=0.1 μM) for 7 days (SN-2414) and non-stimulated (SN-medium) wereadded to lymphoblastoid cell cultures in several concentrations. Theproliferation of the transformed cells was measured by [3H]thymidineuptake (cpm=counts per minute) after 2 days. TABLE 14 Proliferation (cpmafter [3H]thymidine uptake) of lymphoblastoid cell lines in mediumRPMI-1640 Stimulant (dilution) Raji CCL86 Jurkat E6.1 Medium 27617 42536IMM-2414 (0.1 μM) 28708 42779 and under the influence of EA.hy926endothelial cell culture supernatants SN-Medium (1:2) 38556 35917SN-2414 (1:2) 36397 35653 SN-Medium (1:4) 36227 38982 SN-2414 (1:4)28558 34048 SN-Medium (1:8) 30785 40389 SN-2414 (1:8) 22661 39902Comments: Medium = RPMI-1640 completed as specified above IMM-2414 =solution of standard mixture of pentacyclic alkaloids in mediumSN-Medium = supernatant of endothelial cell culture in medium, dilutedwith medium in the ratio given SN-2414 = supernatant of endothelial cellculture stimulated with the standard mixture of pentacyclic alkaloids inmedium for 7 days, diluted with medium in the ratio given

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of low concentration-stimulatedendothelial cell cultures (SN-2414) exhibit only a weak influence on theproliferation of the Raji and Jurkat cells. The proliferation-regulatingfactor binds to interferon-□-antiserum (from sheep) and can be isolatedon sepharose.

As the factor enhances the proliferation of normal B and T lymphocytes,it is assumed that it also increases the release of other factors whichare normally produced by lymphocytes, e.g. interferon-□, variousinterleukins or a granulocyte-macrophage-stimulating-factor. Even if theactivity of the factor is controlled by immunological regulatorycircuits, it can be useful to limit this activity in a specific way. Thetetracyclic alkaloids can be employed for this purpose because of theirdose-dependent inhibition of the activity of the factor.

In addition, experiments were performed using simple derivatives of thepentacyclic alkaloids. From the mixture (IMM-2414) the correspondingcarboxylic acids (IMM-2413) were prepared by alkaline hydrolysis, andalkaloid N-oxides (IMM-2433) by oxidation with hydrogen peroxide.Supernatants of endothelial cell cultures stimulated with IMM-2414,IMM-2413 or IMM-2433 for 7 days (SN-2414, SN-2413, SN-2433) andnon-stimulated endothelial call cultures (SN-medium) were added to Tlymphocyte cultures in several concentrations. It was demonstrated thatthe carboxylic acids had only weak activity and the N-oxides almost none(Table 15). Probably these derivatives, due to their higher polaritycompared with the parent alkaloids, cannot enter the cells. It is alsopossible that the free amine and the methyl ester are essentialpharmacophores. TABLE 15 Proliferation (cpm after [3H]thymidine uptake)of human normal T lymphocytes in medium RPMI-1640 Stimulant (dilution) Tlymphocytes Medium 1724 IMM-2414 (1 μM) 1309 IMM-2413 (1 μM)  876IMM-2433 (1 μM) 1305 and under the influence of EA.hy926 endothelialcell culture supernatants SN-Medium (1:4) 2366 SN-2414 (1:4) 4864SN-2413 (1:4) 2717 SN-2433 (1:4) 2418 SN-Medium (1:8) 2079 SN-2414 (1:8)3103 SN-2413 (1:8) 2850 SN-2433 (1:8) 2327 SN-Medium (1:16) 2062 SN-2414(1:16) 2493 SN-2413 (1:16) 2049 SN-2433 (1:16) 1994 Comments: Medium =RPMI-1640 completed as specified above IMM-2414 = solution of standardmixture of pentacyclic alkaloids in medium IMM-2413 = solution ofcarboxylic acids prepared from the standard mixture of pentacyclicalkaloids in medium IMM-2433 = solution of N-oxides prepared from fromthe standard mixture of pentacyclic alkaloids in medium SN-Medium =supernatant of endothelial cell culture in medium, diluted with mediumin the ratio given SN-2414 = supernatant of endothelial cell culturestimulated with the standard mixture of pentacyclic alkaloids in mediumfor 7 days, diluted with medium in the ratio given SN-2413 = supernatantof endothelial cell culture stimulated with the alkaloid carboxylicacids for 7 days, diluted with medium in the ratio given SN-2433 =supernatant of endothelial cell culture stimulated with the alkaloidN-oxides for 7 days, diluted with medium in the ratio given. All valuesresult of single experiments.

It can be seen that the alkaloids alone do not have an effect comparedto the blank medium. The supernatants of the non-stimulated endothelialcell cultures (SN-medium) already increase the proliferation, thesupernatants of the IMM-2414-stimulated endothelial cell cultures(SN-2414) further enhance this effect. The supernatants of theIMM-2413-stimulated endothelial cell cultures (SN-2413) produce onlyweak effects, the supernatants of the IMM-2433-stimulated endothelialcell cultures (SN-2433) have no effect compared to supernatants of thenon-stimulated endothelial cell cultures (SN-medium).

From these investigations and considerations it is seen that thecomposition of the mixture of isomers cannot be left to chance when acertain action on endothelial cells within a definite time is desired.Elimination from living organisms has to be considered. The differentsolubility of the isomers in water and lipids has also to be consideredwhen a galenic form is developed. In order to obtain a specificinduction of release of the factor the pentacyclic isomers have to beadministered in proportions which are adjusted to the physiologicalequilibrium composition.

In general, RPMI-1640 was used as the culture medium for EA.hy926endothelial cells and lymphocytes, completed with 10% fetal calf serum,2 mM glutamin, 50 units/ml penicillin G and 50 μg/ml streptomycin. ForHUVEC cultures HAM F12 was used, completed with 10% fetal calf serum, 60μg/ml Endothelial Cell Growth Supplement and 100 μg/ml heparin.Supernatants of the endothelial cell cultures, stimulated with oxindolealkaloids for 7 days, were diluted with the medium and added tolymphocyte cultures in several concentrations. Proliferation of thelymphocytes was assayed by [3H]thymidine uptake. Thus, normal cells weretreated with 1 μCi [3H]thymidine for 18 hours, and transformed cellswere treated with 0.5 μCi [3H]thymidine for 5 hours. They were harvestedon nitrocellulose, and radioactivity was measured in a scintillationcounter. Every assay was performed in triplicate.

In vivo Experiments

An extract of Uncaria tomentosa root containing pentacyclic oxindolealkaloids was administered orally to rats and human volunteers, and theeffect on the lymphocyte numbers was studied. The numbers of lymphocytesincreased in patients with a suppressed immune system, whereas thelymphocyte count decreased in patients with a highly activated immunesystem.

EXAMPLES

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) arecultivated for 7 days at 37° C. in HAM F12 nutrient medium which wascompleted with 10% fetal calf serum, 60 μg/ml Endothelial Cell GrowthStimulant and 100 μg/ml heparin. Then the supernatant is taken, filteredsterile, diluted 1:4, and added to cultures of normal human Tlymphocytes. The presence of the factor released from the endothelialcells leads within 5 days to an increase of the proliferation of thelymphocytes by 50%.

Transformed EA.hy926 endothelial cells are cultivated in RPMI-1640nutrient medium which was completed with 10% fetal calf serum, 2 mMglutamine, 50 units/ml penicilline G, and 50 μg/ml streptomycin. Thenthe supernatant is taken, filtered sterile, diluted 1:4, and added tocultures of normal human B lymphocytes. The presence of the factorreleased from the endothelial cells leads within 5 days to an increaseof the proliferation of the lymphocytes by 180%.

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) arecultivated for 7 days at 37° C. in HAM F12 nutrient medium which wascompleted with 10% fetal calf serum, 60 μg/ml Endothelial Cell GrowthStimulant, 100 μg/ml heparin, and which contains pentacyclic oxindolealkaloids (c=0.4 mg/l). Then the supernatant is taken, filtered sterile,diluted 1:4, and added to cultures of normal human T lymphocytes. Thepresence of the factor released from the endothelial cells leads within5 days to an increase of the proliferation of the lymphocytes by 110%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days inRPMI-1640 nutrient medium which was completed with 10% fetal calf serum,2 mM glutamine, 50 units/ml penicilline G, 50 μg/ml streptomycin, andwhich contains pentacyclic oxindole alkaloids (c=0.4 mg/l). Then thesupernatant is taken, filtered sterile, diluted 1:4, and added tocultures of normal human B lymphocytes. The presence of the factorreleased from the endothelial cells leads within 5 days to an increaseof the proliferation of the lymphocytes by 330%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days inRPMI-1640 nutrient medium which was completed with 10% fetal calf serum,2 mM glutamine, 50 units/ml penicilline G, 50 μg/ml streptomycin, andwhich contains pteropodine and isopteropodine (c=0.4 mg/l). Then thesupernatant is taken, filtered sterile, diluted 1:4, and added tocultures of leukemic Jurkat cells (ATCC E6.1). The presence of thefactor released from the endothelial cells leads within 2 days to aninhibition of the proliferation of the lymphocytes by 80%.

Transformed EA.hy926 endothelial cells are cultivated for 7 days in anutrient medium which contains mitraphylline and isomitraphylline (c=0.4mg/l). Then the supernatant is taken, filtered sterile, diluted 1:4, andadded to cultures of highly activated T lymphocytes (lymphoblasts). Thepresence of the factor released from the endothelial cells leads within2 days to an inhibition of the proliferation of the lymphocytes by 40%.

A dose of 1 g/kg bodyweight of an extract from the root of Uncariatomentosa mod. pent. which contains approximately 1% pentacyclicoxindole alkaloids is administered orally to healthy rats. Within 28days a significant (relative and absolute) lymphocytosis develops (in 5male rats from 86.8% to 90.4%, from 8.4 G/l to 8.5 G/l; in 5 female ratsfrom 83.4% to 88.4%, from 4.9 G/l to 5.7 G/l).

A tumor patient whose lymphocyte count is diminished (relative andabsolute lymphopenia, share of lymphocytes: 18% of leucocytes, 1.1 G/l)by chemotherapy (Taxol) is given an extract from the root of Uncariatomentosa in a dose corresponding to 0.6 mg pentacyclic oxindolealkaloids daily. In spite of continued chemotherapy the lymphocytecounts rise significantly within 1 month (share of lymphocytes: 21% ofleucocytes, 1.4 G/l).

A group of patients (n=30) with autoimmune disease received an extractfrom the root of Uncaria tomentosa corresponding to 0.6 mg pentacyclicoxindole alkaloids daily for one year. Initially, they had a mean totalleucocyte count of 8.44 G/l (share of lymphocytes: 21.5%, 1.82 G/l).After half a year of treatment the leucocyte count was 8.66 G/l, thelymphocytes dropped to 18.2%, 1.57 G/l. After one year the leucocytecount was 8.50 G/l, and the lymphocytes remained stable at 18.5%, 1.57G/l.

Transformed EA.hy926 endothelial cells are cultivated for 7 days at 37°C. in a nutrient medium which contains mitraphylline andisomitraphylline (c=0.4 mg/l) or pteropodine and isopteropodine (c=0.4mg/l). Then the supernatant is taken, filtered sterile, diluted (1:8,1:16, 1:32) and added to cultures of normal human B lymphocytes. Thesupernatant which has been obtained from the mitraphylline-stimulatedcells increases the proliferation of the lymphocytes by 260% in alldilutions, whereas the supernatant which has been obtained from thepteropodine-stimulated cells shows a dose-dependent activity (increaseof proliferation by 250%, 170%, and 150%, respectively).

Normal human umbilical vein endothelial cells (HUVEC, ATCC CRL-1730) arecultivated for 7 days at 37° C. in HAM F12 nutrient medium which wascompleted with 10% fetal calf serum, 60 μg/ml Endothelial Cell GrowthStimulant, 100 μg/ml heparin, and which contains pentacyclic oxindolealkaloids (c=0.4 mg/l). Then the supernatant is taken and filteredsterile. 50 μl interferon-□-antiserum (from sheep) in sterile water(c=19000 units/ml) is added per ml of supernatant. The mixture is shakenand incubated for 1 hour at 4° C. Then 25 μl of a 10%protein-A-sepharose suspension is added, incubated for 30 minutes at 4°C. and centrifuged at 2000 g.

From the sediment the factor-antiserum-complex is eluted with a 0.1 Msolution of glycin hydrochloride at pH 2.6.

Extracts from the root of Uncaria tomentosa are tested for the absenceof tetracyclic oxindole alkaloids by High Performance LiquidChromatography (HPLC). A RP-18 (5 μm) column (125×4 mm) is used. Amixture of acetonitrile and 0.01 M phosphate buffer pH 7 (40:60) at aflow of 1.3 ml/min is used as the eluent at 52° C. A relable separationof tetracyclic and pentacyclic oxindole alkaloids is achieved. Detectionis carried out at 247 nm. Only extracts which contain solely pentacyclicoxindole alkaloids are further processed.

Although endothelial cells are not part of the immune system, theypossess the ability to release soluble factors into their environmentwhich affect the behaviour of immune-related cells. It is the object ofthe present invention to effect the release of such a factor whichincreases the proliferation of resting or weakly activated lymphocytesand decreases the proliferation of highly activated lymphocytes andtransformed lymphoblasts without reducing their viability. It is afurther object of this invention to effect the release of this factor bystimulating endothelial cells with pentacyclic oxindole alkaloids. It isyet another object of this invention to limit the release of this factorby the simultaneous administration of tetracyclic oxindole alkaloidswhich act as antagonists. The production and use of this newproliferation-regulating factor are claimed.

1. A method of inducing endothelial cells to release a growth-factorthat is able to regulate proliferation of lymphocytes, said methodcomprising stimulating said endothelial cells with at least onepentacyclic oxindole alkaloid.
 2. The method according to claim 1,wherein said at least one pentacyclic oxindole alkaloid is selected fromthe group consisting of pteropodine, isopteropodine, speciophylline,uncarine F, mitraphylline and isomitraphylline.
 3. The method accordingto claim 1, wherein said endothelial cells are in a living subject andsaid at least one pentacyclic oxindole alkaloid is administered to theliving subject.
 4. The method according to claim 1, wherein saidendothelial cells are cultivated in a nutrient medium containing said atleast one pentacyclic oxindole alkaloid.
 5. A method of producing apreparation containing a growth-factor that is able to regulateproliferation of lymphocytes, said method comprising stimulatingendothelial cells with at least one pentacyclic oxindole alkaloid,whereby said endothelial cells are stimulated to release saidgrowth-factor, and removing the endothelial cells.
 6. The methodaccording to claim 5, wherein, when said endothelial cells arecultivated in a nutrient medium, said at least one pentacyclic oxindolealkaloid is added to said nutrient medium to induce a release of saidgrowth-factor into said nutrient medium, and removing the endothelialcells from said nutrient medium.
 7. The method according to claim 5,wherein said at least one pentacyclic oxindole alkaloid is selected fromthe group consisting of pteropodine, isopteropodine, speciophylline,uncarine F, mitraphylline and isomitraphylline.